Myotonic dystrophy associated expanded CUG repeat muscleblind positive ribonuclear foci are not toxic to Drosophila

Abstract

Myotonic dystrophy type 1 is an autosomal dominant disorder associated with the expansion of a CTG repeat in the 3' untranslated region (UTR) of the DMPK gene. Recent data suggest that pathogenesis is predominantly mediated by a gain of function of the mutant transcript. In patients, these expanded CUG repeat-containing transcripts are sequestered into ribonuclear foci that also contain the muscleblind-like proteins. To provide further insights into muscleblind function and the pathogenesis of myotonic dystrophy, we generated Drosophila incorporating CTG repeats in the 3'-UTR of a reporter gene. As in patients, expanded CUG repeats form discrete ribonuclear foci in Drosophila muscle cells that co-localize with muscleblind. Unexpectedly, however, foci are not observed in all cell types and muscleblind is neither necessary nor sufficient for their formation. The foci are dynamic transient structures with short half-lifes that do not co-localize with the proteasome, suggesting they are unlikely to contain mis-folded proteins. However, they do co-localize with non-A, the human orthologs of which are implicated in both RNA splicing and attachment of dsRNA to the nuclear matrix. Muscleblind is also revealed as having a previously unrecognized role in stabilizing CUG transcripts. Most interestingly, Drosophila expressing (CUG)162 repeats has no detectable pathological phenotype suggesting that in contrast to expanded polyglutamine-containing proteins, neither the expanded CUG repeat RNA nor the ribonuclear foci are directly toxic.

Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy

“…The (CTG) 5 transcripts are not retained, so overexpression may represent an accelerated version of this process. Nevertheless, the present findings, our previous findings in a myoblast cell culture model 24 and, more recently, Drosophila melanogaster models 25 and a study showing that RNA foci formation and splicing defects are separable 26 highlight the need to reexamine the role of RNA-protein sequestration in myotonic dystrophy pathogenesis.…”

Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy

“…Second, in a C2C12 cell culture model, foci formation was not correlated with the defect in myoblast differentiation observed in DM cultures, suggesting that foci formation was not sufficient to disrupt myogenic differentiation (Amack and Mahadevan, 2001;Furling et al, 2001). Similarly, in a transgenic Drosophila melanogaster model expressing 162 CTG repeats, the presence of CUG foci colocalizing with Drosophila muscleblind protein did not cause muscle defects or alterations in locomotive activity (Houseley et al, 2005). Third, although the foci are associated with DM in muscle, DM tissues contain small numbers of foci (1-3) relative to cultured myoblasts (>20) and it is unknown whether these are sufficient to quantitatively inactivate a large fraction of cellular MBNL protein (Davis et al, 1997;Mankodi et al, 2003;Mankodi et al, 2001).…”

Misregulation of Alternative Splicing Causes Pathogenesis in Myotonic Dystrophy

“…The finding that CAG-repeat RNA forms foci, colocalizes with MBNL but does not alter splicing strongly suggest that foci formation and the potential to sequester MBNL alone is not sufficient for misregulated alternative splicing. Additional support for the inconsistency between toxicity and foci formation comes from a recent report showing that expression of 162 CTG repeats in the 3′ UTR of a reporter gene formed foci in Drosophila tissues without inducing pathology, suggesting that foci formation was not toxic to Drosophila (Houseley et al 2005). Similarly, foci formation by RNAs containing only CUG repeats is not sufficient to induce muscle-differentiation defects in the C2C12 cell line (Amack and Mahadevan 2001).…”